The field of this invention relates to radionuclides employed in diagnostic nuclear medicine, particularly the gamma emitting diagnostic agents for imaging by scintillation scanning. More particularly, this invention is concerned with the radionuclide technetium-99m which when chelated by certain ligands produces clinically useful radiopharmaceuticals. Tc-99m has nearly ideal physical properties for scintigraphic imaging techniques and, despite limited specificity of previously available ligands when complexed to Tc-99m, is the most frequently used radionuclide for imaging in diagnostic nuclear medicine. For most in vivo uses, the metal, which is available as pertechnetate, must be complexed in a reduced form by ligands which produce complexes that are stable in aqueous solutions and body fluids and, which, if properly structured, display specific biodistributions.
The chemical synthesis of ligands that form Tc-99m complexes with improved properties has been hindered by several factors. Pertechnetate (TcO.sub.4.sup.- -99m) is readily available but only in extreme dilutions. For example, the commonly employed molybdenum-99 (Mo-99) generator, which produces Tc-99m as a radioactive decay product, is in the form of an alumina column that is eluted with aqueous sodium chloride to produce the pertechnetate solutions in the range of 10.sup.-6 to 10.sup.-9 molar concentration. Such dilutions make it very difficult to study the reactions involved in chelate formation, or to chemically and structurally characterize the resulting coordination complexes. Furthermore, the chemistry of technetium itself is poorly understood.
Although Tc-99m chelates have provided a variety of clinically useful radiopharmaceuticals for diagnostic imaging, producing suitable Tc-99m-compounds with high physiological and organ selectivity is a complex task. Certain constraints limit the number of chelating ligands that would be appropriate for formulating new diagnostic agents labeled with Tc-99m. Since Tc-99m has only a 6 hr half-life, stable Tc-99m chelates must be produced rapidly (under sterile, apyrogenic conditions) in high yields in neutral aqueous solutions so that they may be readily available to all clinical Nuclear Medicine laboratories. In addition, the ligands must show no adverse toxicity for routine use as diagnostic agents in human studies.
All Tc-99m-chelates so far approved by the FDA for routine use in Nuclear Medicine have overall negative or positive charges. Most ligands used to complex Tc-99m in high yields in aqueous media contain O, N, S, or P donor atoms. These complexes are very useful for evaluating the functional status of human tissues or organs in which a charged species would be advantageous (i.e., kidney filtration, hepatobiliary function by the anionic or cationic clearance pathway, brain imaging involving the breakdown of the blood-brain-barrier (BBB), cation localization in heart muscle, bone uptake or phosphonate chelates, etc.) Charged hydrophobic complexes are not now routinely available to evaluate biological functions where the Tc-99m-chelate must passively diffuse across cell walls or the intact BBB. The most clinically applicable agent for these latter applications would be a stable neutral-hydrophobic Tc-99m chelate that would lead to the design of a series of new diagnostic radiopharmaceuticals.
An area where a suitable neutral-hydrophobic Tc-99m-chelate would find immediate diagnostic application is to assess regional cerebral blood flow (rCBF) in humans. The need for this type of Tc-99m labeled agent has been recognized for some time. See Oldendorf, J. Nucl. Med., 19, 1182, Letters to Ed (1978). As stated by Dr. Oldendorf: "I believe that a concerted effort should be made to develop a Tc-99m labeled compound which is sufficiently lipid-soluble that it would undergo complete clearance in one pass through the brain".
Compounds labeled with short-lived positron emitters (e.g., C-11, O-15 and F-18) have been used to successfully delineate rCBF in normal and diseased brain tissue using Positron Emission Transaxial Tomography in a limited number of Nuclear Medicine laboratories. The costs to produce these compounds (including an on-site medical cyclotron) are prohibitive for wide spread use. If we are to witness the performance of such studies in the daily practice of medicine, the development of agents capable of rCBF assessment but labeled with more available and less costly radionuclides emitting a single photon (i.e., a .gamma.-ray), such as Tc-99m is highly desirable.
Two types of single photon emitting diagnostic agents have been used to assess brain blood flow patterns in humans. The first is best exemplified by Xe-133, an inert noble gas, that passively diffuses across the BBB and clears the tissue at a rate proportional to the blood flow through that tissue. The pattern of rCBF is determined by following the rate of Xe-133 clearance as a function of time at different sites of the brain using either a specialized Single Photon Emission Computed Tomographic (SPECT) instrument (1) or a commercial multidetector system (2). The second class of compounds include those that passively diffuse across the BBB (with a high extraction efficiency) into the brain and become trapped in the brain tissue. The trapping allows time for determination of rCBF patterns by more conventional SPECT imaging devices. The two most widely used single photon brain perfusion agents of this latter type are: I-123-N,N,N'-trimethyl-N-(2-OH-3-methyl-5-iodobenzyl)-1,3-propanediamine,( I-123-HIPDM) (3) and I-123-Iodoamphetamine, (I-123-IMP) (4,5). Because Tc-99m has superior imaging properties and is more readily available and less expensive, any new Tc-99m labeled compounds with similar or even better capabilities for brain uptake (and/or washout) as Xe-133, I-123-IMP or I-123-HIPDM would find widespread clinical applicability.
Besides rCBF studies these neutral-lipophilic Tc-99m-chelates would also be desirable in imaging the lungs. I-123-iodoantipyrene is a hydrophobic compound that distributes following injection throughout the lung water and has been used to assess extravascular lung water (EVLW) in normal and disease states (6). This agent passively diffuses into the lung parenchyma and is washed out by pulmonary blood flow. Lung imaging has also been performed using I-123-IMP by J. Tonya, et al., (7). I-123-IMP is taken up by lung tissue and slowly released, presumably due to its binding to intracellular low specificity, - high capacity endothelial amine receptors. Clearly, Tc-99m agents that exhibit properties similar to I-123-iodoantipyrene would have value for measuring regional EVLW imaging (particularly in patients with the acute respiratory distress syndrome (6)). Likewise, a Tc-99m-compound that binds to amine receptors in the lung would be useful for assessing what Tonya et al. (7) describe as "metabolic lung imaging".
Imaging of heart muscle in patients with myocardial damage using fatty acids or fatty acid analogues labeled with positron emitters (8) or I-123 has shown promise as a diagnostic tool (9). As indicated earlier, the high cost of producing compounds labeled with positron emitters precludes their widespread availability in the foreseeable future.
Under normoxic conditions the energy requirements of heart muscle are met by oxidation of fatty acids and, therefore, the extraction of free fatty acids by the normal myocardium is high. In areas of heart muscle damage where local pO.sub.2 is decreased (eq. ischemia) fatty acid oxidation (i.e., .beta.-oxidation) and free fatty acid uptake is decreased (10). Regional fatty acid metabolism could be measured by determining the rate of clearance of the labeled fatty acid from the myocardium. Alternatively, a fatty acid analogue that enters the metabolic pathway, undergoes partial metabolism, and whose radioactive label is trapped within the myocardium would also reflect regional metabolic activity (i.e., similar to 18-F-fluorodeoxyglucose for brain metabolic images (11)). The localized trapped activity can then be imaged (12). Fatty acid analogues labeled in various manners with I-123 are taken up by normal heart muscle and depending upon the structure of the specific compound will either clear rapidly following intracellular .beta.-oxidation (similar to C-11 labeled fatty acids) or can be structurally modified so they will be trapped by the myocardial tissue (13,14). I-123 can be attached directly to the .omega.-end (i.e., the end opposite the carboxylic acid group) of the alkyl-chain or attached to the .omega.-end of the alkyl chain by means of a I-123-phenyl group. Both types of derivatives have been successfully used as myocardial imaging agents (15,16). The fact that good myocardial uptake is observed with fatty acid analogues where the bulky-hydrophobic I-123-phenyl group is attached to the .omega.-end indicates that other lipophilic groups similarly attached will have little effect on their extraction by normal heart muscle. Karesh, et al., (17) and Schneider et al., (18) failed in their attempts to produce a Tc-99m-fatty acid analogue that localizes in heart muscle. Both investigators attached ligands to the .omega.-ends of fatty acid analogues that formed negatively charged Tc-99m-chelates and concluded that the charged chelate at the end of the alkyl chain prevented their intracellular transport. It is expected that a neutral-hydrophobic Tc-99m chelate attached to the .omega.-end of fatty acid analogues will not prevent normal myocardial cell uptake (viz., these types of compounds would be structurally similar to the I-123-phenyl-.omega.-fatty acid analogues).
At least three neutral Tc-99m-complexes have been prepared and reported. See, Burns, et al., J. Nucl. Med., 20, 641, 1979. Kramer, et al., Proc. 4th Int. Symp. on Radiopharmaceutical Chemistry, Julich, Germany, Aug. 23-27, 1982, p. 323-324. Yokoyama, et al., J. Nucl. Med., 17, 816-19 (1976). None of these Tc-99m complexes have been established for routine diagnostic use. These neutral ligands have not met all of the needed requirements; for example, the neutral complexes of Yokoyama, et al. are extremely difficult to derivatize and use --SH groups for complexation with Tc-99m as does the neutral complex of Burns et al. Ligands utilizing --SH groups for chelation of Tc-99m form stable complexes but are difficult to store. The neutral chelate of Kramer et al., does not have desirable stability characteristics. As far as known, none of these ligands show a high extraction efficiency by the brain.
Ligands that employ only N-atoms for chelation of Tc-99m have been shown to readily form complexes in aqueous media. Tetraaza ligands and in particular macrocyclic tetraaza ligands, form very stable Tc-99m complexes. See, Troutner, et al., J. Nucl. Med., 21, 443-448 (1980), which describes the complexing of TcO.sub.4.sup.- -99m with the macrocyclic tetraaza ligand, cyclam. As far as is known, the only published study showing full characterization of such a macrocyclic ligand complex of Tc was published in 1981 by Zuckman, et al., Inorg. Chem., 20, 2386-2389. The complex with cyclam (1,4,8,11-tetraazacyclotetradecane) under reducing conditions was shown to produce a TcO.sub.2.sup.+1 core, and a resulting complex had a charge of +1.
As far as is known, the ligand specifically referred to in this disclosure as propylene amine oxime (PnAO) has not been employed to prepare a complex with Tc-99m. This ligand is 2,2'-(1,3-diaminopropane) bis (2-methyl-3-butanone) dioxime and is henceforth referred to as PnAO. Methods for preparing PnAO are well known as is its use in complexing metal ions. See, for example, Vassian, et al., (19). Complexation of PnAO to metal ions is by the 4 N-atoms and results in the liquid forming a cyclical structure around the chelated metal (20). The structural formula of PnAO is: ##STR1##
The corresponding ethylene amine oxime (EnAO) is 2,2'-(diaminoethane) bis(2-methyl-3-butanone) dioxime, and the corresponding n-butylene amine oxime (BnAO) is 2,2'-(1,4-diaminobutane) bis(2-methyl-3-butanone) dioxime.